The authors are members of the Analytical and Environmental Research Group at Dublin City University. Dr Regan’s research interests include the development of nanoparticles for biofouling prevention and novel separation systems

Nanoparticles in Anti-Microbial Materials

Use and Characterisation

By Fiona Regan, James Chapman, Timothy Sullivan

The Royal Society of Chemistry

Copyright © 2012 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-159-1

Contents

Preface, iv,
Chapter 1 Nanoparticles: What Are They?, 1,
Chapter 2 Microbial Impacts on Surfaces, 30,
Chapter 3 Applications of Nanoparticles, 69,
Chapter 4 Characterisation of Materials using Quantitative Approaches, 94,
Chapter 5 Visualisation of Nano-anti-microbial Materials, 114,
Chapter 6 Biological Methods for Characterisation of Nano-anti-microbial Materials, 153,
Chapter 7 Molecular Biological Techniques, 194,
Chapter 8 Conclusions, 228,
Subject Index, 231,

CHAPTER 1

Nanoparticles: What are They?

1.1 Introduction

Although it was seldom reported in much of the literature before the 1990s, nanotechnology has recently taken the world of science by storm. Today, nanotechnology has taken a multi-faceted scientific route, with exploration undertaken in a breadth of disciplines in science ranging from, but not limited to, material science, chemistry, biology and physics to name but a few. The capability of synthesising and manipulating materials at the nanoscale interests the scientific community, the modern technological world and even the press. This burgeoning interest is amplified by the unique physical and chemical properties that nanomaterials exhibit and the promise they hold for use in future technologies such as those requiring unique optical, electrical and magnetic properties and, the focus of this book, anti-microbial activity.

1.2 Nano, the Beginning to the Present

One of the earliest reports of nanoscale particles related to gold. Faraday demonstrated the preparation of colloidal gold, which he named ‘divided metals’. His account, dated 2nd April 1856, called the particles he made ‘the divided state of gold’, solutions of which, remarkably, can still be found in the Royal Institution in Mayfair, London, UK.

Later in 1890, the early German microbiologist Robert Koch proved that compounds incorporating gold inhibited the growth of bacteria, the discovery of which led to his Nobel Prize for medicine in 1905. Indeed the use of gold in medicine is not new and indications of the use of gold for medicinal purposes can be found throughout history. In India, for example, gold has been prepared for memory prescriptions known as Sarawatharishtam. In China, a gold coin was used in cooking rice, a practice said to help replenish a deficit of gold in the body.

However, the science of nanoscale objects was not discussed until much later in the history books, not until Richard Feynman gave a talk entitled There’s Plenty of Room at the Bottom in 1959 at an American Physical Society lecture. During his talk he stated, ‘The principles of physics, as far as I can see, do not speak against the possibility of manoeuvring things atom by atom’. This, in a way, was the first suggestion of a bottom-up approach to nanomaterial synthesis. Richard Feynman went on to state ‘… it is interesting that it would be, in principle, possible for a physicist to synthesize any chemical substance that the chemist writes down. Give the orders and the physicist synthesizes it. How? Put atoms down where the chemists say, and so you make the substance. The problems of chemistry and biology can be greatly helped if our ability to see what we are doing, and to do the things on an atomic level, is ultimately developed – a development which I think cannot be avoided’.

However, it was not until 1981 that tools became available for probing such a hypothesis, with the advent of the scanning tunnelling microscope (STM). This tool enabled unprecedented visualisation and manipulation of materials at the nanoscale. Ultimately, such ability has led to the current interest and growth of nanotechnology research, which is starting to come to fruition as new nanotechnological products reach the marketplace and consumer in the immediate future.

1.3 Defining the Nanodimension

For the purpose of this book we limit the discussion of nanomaterials defined by a minimum of two dimensions less than 100 nm. A current trend of material within this scope can be principally traced to work by Luis Brus in the 1980s in which he postulated that the band gap of a simple direct band gap semiconductor should be dependent on its size once its dimensions were smaller than the Bohr radius.

As a final point, as the research field of nanotechnology and nanomaterials has evolved rapidly with much ambiguity with regard to terminology, it is prudent to introduce some fundamental definitions:

• Colloid – a stable liquid phase containing particles in the range of 1–1000 nm. Particles in the 1–1000 nm range have the ability to be colloidal particles, as shown in Figure 1.1;

• Nanoparticle – a solid particle in the 1–1000 nm range which may either refer to a non-crystalline particle, an aggregate of crystallites or even a single crystallite;

• Quantum dot – a particle that exhibits a size quantisation effect in at least one dimension;

• Nanomaterial – any solid material that has a nanometre dimension. In summary, three dimensions = particles, two dimensions = thin films, one dimension = nanowire.

1.4 Physical Chemistry of Nanoparticles

A predominant feature of nanomaterials relates to the disproportionate influence of the surface area to volume ratio as materials enter the nanoscale dimension. Nanomaterials typically exhibit a high surface area to volume ratio, which has many interesting effects on the subsequent behaviour of these materials. As the radius r of a spherical particle decreases, the surface/volume ratio 3/r and the proportion of the constituent atoms at the surface both increase. The stable interatomic bonding that exists in large crystals is not satisfied for atoms at the surface and these therefore become more mobile and reactive, so that the overall properties of the nanomaterial become dominated by surface chemistry. The nanoparticles discussed below will be based on a metallic model, but most of the characteristics described will also apply to non- metal based materials. This will be addressed in the following contexts:

1. in aqueous environments;

2. in a two-phase mixture (i.e. polymer-metal or ceramic metal mixture);

3. on an insulating substrate surface, as a discontinuous (island) metal thin film (DMTF) of nanoparticles.

In cases 1 and 2, the nanoparticles are usually modelled on spherical particles, but particle shape can interject for DMTF. A classical nucleation theory will form the basis of formation and growth of nanoparticles but prediction of critical nucleus sizes in the sub-nanometre range is clearly inconsistent with the classical model’s use of bulk thermodynamic properties. For nuclei containing only a few atoms, as is typically used in all three of these systems, the atomistic nucleation theory is necessary.

1.4.1 Structure

Nanoparticles can exist as perfect crystals since impurities and lattice defects can migrate to the surface in relatively short periods of time. Debye-Scherrer broadening of electron diffraction patterns provides a means of determining nanocrystallite sizes, with their radii giving specific lattice spacing, discussed later in this book. Nanoparticles in aqueous environments are usually assumed to be spherical at concentrations sufficiently below a percolation threshold where the particle contacts and coalescence can be ignored. However, in a liquid environment the size and shape (i.e. cubes, prisms, rods, spheres) of the nanoparticle can be controlled through variation of the precipitation condition.

In the absence of other conditions, the minimum energy configuration for a nanoparticle with bulk and surface energies has been shown to be a sphere, but the equilibrium minimum energy shape of a charged particle is actually an ellipsoid of rotation. Regarding the DMTF, the nanoparticle islands appear to be slightly prolate in deposition as r2 is proportional to time (or mass) not r3 as expected for quasi-spherical growth. So the dominant particle shape is oblate, with possible causes being electrostatic or substrate adatom capture with insufficient thermal energy to reach spherical equilibrium. Another factor in determining nanoparticle shape may be related to the degree of crystallisation and the relatively weak binding of the surface atoms, which in particular, permits rapid motion and continual abrupt crystallographic reconstructions in the size range of 1–10 nm.

1.4.2 Electrical Properties

If the surface atoms are characterised by incompletely satisfied chemical bonds and the surface can be considered to be disordered, then this disorder extends to the crystal interior as dimensions decrease. If metallic conductivity is associated with band structure of the regular crystal, the question arises as to whether metallic properties can be maintained at nanoscale dimensions. Kreibig applied a different criterion, the experimental observations of dielectric absorption in glass containing Ag or Au nanoparticles, concluding that the cluster-solid state transition occurs at ~500 atoms/particle (i.e. ~2.5 nm diameter).

More recently, the electronic properties of metal nanoparticles have been under intense investigation, with a view to decreasing electronic device size and making features ever smaller within the nanoscale. The application of individual transistors, electrometers, chemical sensing, anti-microbial agents, wireless logic and memory have all been demonstrated in principle, albeit to a broad degree at this point in time.

1.4.3 Catalytic Properties

Catalysts are used to speed up chemical reactions, typically by one of two mechanisms:

1. provision of a new reaction path of lower activation energy;

2. provision of a surface to which the chemical reactants can adhere and react more readily than, for example, in the gas phase.

The rapid advances in nanotechnology have spawned many new innovations in nanoparticle catalysis, involving their use in biomedical applications, as seeds for vapour–liquid–solid (VLS) growth processes in chemical vapour deposition (CVD) of both carbon nanotubes (CNTs) and nanowires and the use of carbon nanoparticles and CNTs as support structures for nanoparticle catalysts, ensuring maximum active surface exposure.

1.4.4 Mechanical Properties

The effects of nanomaterials on the mechanical properties of thin films have been studied extensively, and the production of thin films incorporating nanomaterials exhibiting various desirable properties has led to a rapid improvement in such materials. Generally these films show enhanced proper- ties as a function of decreasing nanomaterial size. At a fundamental level, this improvement makes sense owing to the relative lack of grain boundaries and defects in nanocrystalline structures. For a metallic-based thin film, yield strength is proportional to r1/2 (the Hall–Pecht relation) and granular ceramic-metals display discontinuous metallic percolation paths above this threshold.

In more recent applications, the effective modelling of nanocomposites at the nanoscale and the mechanical properties of the nanoparticles themselves must be discussed. The obstacles to making such measurements on the nanometre scale are apparent but progress is still possible.

1.4.5 Surface Plasmon Resonance

Surface plasmons (SP) are oscillations of electron density in the conduction band that occur at the metal/dielectric interface, exhibited at the metal/water and the metal/air interfaces. SP propagate evanescently in waveform, and any wave can have its amplitude enhanced with a wave of light with the same resonant wavelength applied to it. Excitation of SP with electromagnetic radiation is termed SPR. Stronger SP have been known to be associated with the roughness of the metal nanoparticle surface. All of these properties are unique to the metal nanoparticle which can be brought about in synthesis.

1.4.6 Anti-microbial Properties

The anti-microbial potential of nanomaterials has gone unrecognised throughout history due to the distinct lack of understanding of the mode of toxicity towards certain organisms, although the first reports of anti-microbial materials based on metals date back to Cyrus the Great, King of Persia, who establisheda board of health and medical dispensary for his citizens. The ability to recognise this potential has, of course, only become possible through the advancement of modern science, where advances in other research fields have contributed techniques capable of analysing and visualising nanomaterials.

Silver has been used for centuries to prevent and treat a variety of diseases, and Davies and Etris have suggested that silver may be linked to man’s earliest attempt to improve his environment. Only now has it become known that silver has a positive influence on the reduction of microbial activity, whose mechanisms are still not fully understood, but can be summarised in the following manner:

• destruction of the organism by oxidation catalysed by silver;

• disruption of the electron transfer in bacteria through the monovalent silver species and preventing the unravelling of DNA in viruses through the substitution of hydrogen ions in the monovalent form;

• destruction of bacteria and viruses through the bivalent and trivalent silver ion form.

Silver holds unique properties over all the other metals, especially when introduced with oxygen. Oxygen in the air is adsorbed on to the surface of silver as atomic oxygen and because atomic oxygen fits into the octahedral holes of silver, oxygen accumulates within the bulk of silver. This stored octahedral oxygen significantly contributes to the catalytic activity and thus oxidative power of silver. It was not until 1986 that a filed patent in the USA covered the catalytic activity of silver in an aqueous media for sanitation of water.

1.4.6.1 Silver as an Anti-microbial Agent

Anecdotal evidence of the health benefits of silver have existed for centuries. Since ancient times, silver vessels have been used in Mexico (incidentally, the world’s largest producer of silver) to keep water and milk ‘sweet’. Following this, Pliny the Elder, reported in his book Natural History (78 A.D.), Book XXXIII, Section XXXV, that silver ‘… has healing properties as an ingredient in plasters, being extremely effective in causing wounds to heal …’. These books comprise one of the only original natural history books from the Roman Empire to survive through to modern day science. World War II however saw a regress in the use of silver when it was discovered that silver plates in the skull caused a toxic reaction at the blood–brain barrier.

Silver in its monovalent state has a high affinity for a sulfhydryl group exposed to bacteria or viruses. This has been seen to inhibit hydrogen transfer in energy transfer systems. The medicinal value of silver has long been recognised where silver nitrate was mentioned in a Roman pharmacopoeia in 69 BC Silver nitrate is, however, corrosive to tissue and draws chloride ions out of the body’s fluids. Thus, less aggressive silver compounds are more suitable for medical purposes. Silver thiosulfate coated in silica gel microspheres was developed by Okada and Suehiro Here the authors incorporated silver in a silica gel, which in turn slowly releases silver over a long term without being immediately precipitated by any nearby chloride species and thus provides long lasting bactericidal action.

1.4.6.2 Emerging Nanoparticles

In recent works by Chapman et al., gallium, germanium and selenium metal nanoparticles were shown to be effective as antifouling nanoparticles. The nanoparticles were synthesised using a polyol reduction, doped into sol–gels and then tested for anti-microbial activity. Furthermore, the authors report that gallium had shown improved antifouling and indeed antimicrobial responses in pure culture assays with E. coli.

New amphiphilic cobalt-meditated and silver-loaded nanoparticles have also been investigated and characterised in a paper by Bryaskova et al. These authors reported that the anti-microbial activity of the silver loaded micelles when tested against P. aeruginosa, S. aureus, E. coli and B. subtilis showed an anti-microbial effect against S. aureus, E. coli and P.aeroginosa at concentra tions of 1.44 µg mL-1.

Other metal oxide nanoparticles are also known to possess potent antimicrobial properties. For instance aluminium oxide nanoparticles are already found abundantly in personal care products. Yamamoto et al. investigated the cytotoxicity of metal particles (Al2O3, titanium dioxide, zirconium oxide) on murine fibroblasts and murine monocyte macrophages. The authors reported the size, shape and overall surface area of the nanoparticles and also compared the toxicity behaviour to the bulk and nano produced materials. Similarly, Sadiq et al. have produced aluminium metal nanoparticles and assessed biological activity using the Gram-negative bacteria, E. coli. The aluminium nanoparticles showed a mild microbial growth inhibitory effect, probably due to disruption of the cell membrane owing to the generation of reactive oxygen species by the particle. The aluminium was also reported to act as a free-radical scavenger, highlighting further emerging uses.

Nanosensors are in early stages of development and have the ability to detect bacteria alone and among other contaminants, such as mycotoxins. This allows a more frequent testing regime to be set up at a much lower cost.

(Continues…)Excerpted from Nanoparticles in Anti-Microbial Materials by Fiona Regan, James Chapman, Timothy Sullivan. Copyright © 2012 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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